CN110201191A - A kind of functional protein and the compound of cyanine dye molecule and its preparation method and application - Google Patents
A kind of functional protein and the compound of cyanine dye molecule and its preparation method and application Download PDFInfo
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- CN110201191A CN110201191A CN201910617372.1A CN201910617372A CN110201191A CN 110201191 A CN110201191 A CN 110201191A CN 201910617372 A CN201910617372 A CN 201910617372A CN 110201191 A CN110201191 A CN 110201191A
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- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
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- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0032—Methine dyes, e.g. cyanine dyes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0032—Methine dyes, e.g. cyanine dyes
- A61K49/0034—Indocyanine green, i.e. ICG, cardiogreen
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- A—HUMAN NECESSITIES
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- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0056—Peptides, proteins, polyamino acids
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- A—HUMAN NECESSITIES
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- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
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- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
- A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
- A61K49/0089—Particulate, powder, adsorbate, bead, sphere
- A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
- A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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Abstract
The present invention provides the compound of a kind of functional protein and cyanine dye molecule, it is to pass through the compound being controllably self-assembly of by cyanine dye molecule and functional protein, the compound outer layer is the functional protein, and the cyanine dye molecule is enclosed with inside the functional protein.Compound of the present invention is obviously improved compared to existing two region probe of near-infrared with luminous and internal behavior, has the imaging effect significantly improved in 2nd area of near-infrared, can be applied to living body fluorescent imaging and molecular imaging.Application the present invention also provides the method for preparing the compound and the compound in 2nd area of near-infrared as fluorescent molecules imaging imaging agent.
Description
Technical field
The invention belongs near infrared imaging probe technique fields, and in particular to it is a kind of it is novel based on functional protein and
The compound of cyanine dye, and preparation method thereof and as two area's Complex Probes of near-infrared application.
Background technique
The development of bio-imaging technology at present is greatly enriched the means that researcher explores bioprocess, mechanism, and glimmering
Light imaging (fluorescence imaging) has many advantages, such as fast image taking speed, high sensitivity, can carry out targeting design, wide
It is general to be applied in cell, tissue and living imaging.Bio-imaging mainly by specific fluorescence probe, selects suitable imaging to set
Standby (including sensitive wide field from visible region near infrared region or microscopy apparatus), to study the side of biological process and information
Method.The key of Imaging-PAM is to come the fine structure of discernable cell or tissue, Jin Erfen with enough signal contrasts
Analyse the information such as the feature of specific region, the expression of state or even specific molecular, distribution.Imaging-PAM also has simultaneously to be divided
The advantages that resolution height, lossless detection, therefore be widely used in exploring disease pathogenesis, clinical disease detects, and navigate hand in art
The fields such as art.
As we state in patent document CN107796796A, bioluminescence imaging still face in terms of living imaging
Face huge bottleneck and challenge, there are two aspects for main cause: one is there are the spontaneous glimmering of visible light wave range for organism itself
Light causes the background of living imaging to increase, and resolution ratio substantially reduces；Secondly since there is strong penetrating organism for photon
Scattering process, therefore deep living body fluorescent imaging can not be compared.The method for solving the problems, such as this be exactly select launch wavelength compared with
Long one area of near-infrared (750-900 nanometers) the even fluorescent material in 2nd area of near-infrared (1000-1700 nanometer) carry out it is biological at
Picture.
Although the fluorescent emission hangover that we and other researchers realize the cyanine dyes such as IRDye 800CW is applied to
(900-1500 nanometers) of 2nd area of near-infrared imaging, so that it be made to may be directly applied to near-infrared as a kind of ready-made commercial dyes
Two area's living imagings.But two area's luminous efficiency of near-infrared of cyanine dye is relatively low, be easy photobleaching, living body stability compared with
Difference, vivo applications are easier to the weakness of diffusion, cause its imaging window shorter.Therefore, it is desirable to the promotion near-infrared two of real meaning
The low background, excellent penetration depth and lower biological tissue scatters of living imaging are realized in the clinical application of area's imaging technique,
There is an urgent need to luminous mass height, circulation time in vivo length and stable two region probes of near-infrared for we.
Summary of the invention
It is compound that primary and foremost purpose of the invention is to provide a kind of two region probe of near-infrared that luminous and internal behavior is obviously improved
Object can be applied to living body fluorescent imaging and molecular imaging.
It is another object of the present invention to provide the methods for preparing the probe complex.
It is yet a further object of the present invention to provide the probe complex in 2nd area of near-infrared as fluorescent molecules imaging
The application of imaging agent.
To achieve the goals above, the invention adopts the following technical scheme:
Firstly, provide the compound of a kind of functional protein and cyanine dye molecule, it be by cyanine dye molecule and
For functional protein by the compound being controllably self-assembly of, the compound outer layer is the functional protein,
The cyanine dye molecule is enclosed with inside the functional protein.
In compound of the present invention, the cyanine dye molecule is that have spreading out for any one following skeleton structure
Biology:
Cyanine dye molecule further preferred ICG or IR-125 with the skeleton 1；
Cyanine dye molecule further preferred IR-820, IR-830 or Cy7.5 with the skeleton 2；
Cyanine dye molecule further preferred DiR or HITCI with the skeleton 3；
The further preferred IRDye800cw, IR-783 of cyanine dye molecule, IR12-N3, Cy7 with the skeleton 4,
IR-775 or IR-780；
In compound of the present invention, most preferred cyanine dye molecule is IR-783, IR-12N3 or ICG.
In compound of the present invention, the functional protein can be existing a variety of with physiological function
Protein, including catalytic protein, transport protein, immune protein, regulatory protein etc..Currently preferred functional protein packet
It includes: albumin, glycoprotein, lipoprotein, globulin or other antibody；The further preferred Erbitux of other antibody
(Erbitux), anti-CD31 antibody or herceptin (herceptin) etc..
In a kind of currently preferred embodiment, the compound is by the cyanine dye molecule and albumin
By what is be controllably self-assembly of, the compound outer layer is albumin, and the flower is enclosed with inside the albumin
Green dye molecule；The cyanine dye molecule is IR-783, IR-12N3 or ICG.The albumin can be human seralbumin
Albumen or animal serum albumin.
On this basis, the present invention further provides the methods for preparing the compound, comprising:
1) solution A containing the functional protein is mixed with the solution B containing the cyanine dye molecule,
The control functional protein and the cyanine dye molecule reach the molar ratio of 1:0.5~1, and it is small that incubation 4-24 is blended
When；Intermediate product is obtained after purification；
2) 1) gained intermediate product 5~15 minutes are heated at 55 DEG C~70 DEG C, obtain the compound.
In preparation method of the invention, after being blended by special ratios, the cyanine dye molecule can be by the function
It can coat to form nano particle to property protein stabilization；Further heat treatment obtains stable compound.The compound is whole
Body is in the liquid of clear.
In order to increase the nanoparticle size that the compound is formed, in preferred embodiments of the present invention, 1) solution A described in
Further contain disulfide bond reducing agent and/or amino crosslinker.The disulfide bond reducing agent and the functional protein
Ratio control in 40-60:1；The ratio of the amino crosslinker and the functional protein is controlled in 15~25:1.
The disulfide bond reducing agent can interrupt the cystine linkage in the functional protein, promote cyanine dye molecule by preferably
Package；The amino crosslinker can be crosslinked the amino in the functional protein and form Schiff base structure.Finally may be used
The composite particles of 50-100 nano-scale are formed to realize that multiple functional proteins fit together.
In the solution of the present invention, the disulfide bond reducing agent can be to be ground suitable for cell biology in the prior art
The various disulfide bond reducing agents studied carefully, such as 2 mercapto ethanol (beta-ME), dithiothreitol (DTT) (DTT) or reduced glutathione
(GSH), the preferred GSH of the present invention；The amino crosslinker can be the various friendships for being suitable for protein cross in the prior art
Join agent, the present invention preferably has the bifunctionality crosslinking agent of high reaction activity, most preferably glutaraldehyde or double amber imide with amino
Ester (double NHS).
In the further preferred scheme of the present invention, 1) purifying described in be washed with the centrifugation filter membrane of 30k it is not anti-to remove
The molecules such as the disulfide bond reducing agent and/or amino crosslinker answered.
The present inventor is found through experiments that, in the preparation method, 1) functional protein described in is in mixed solution
In concentration the size of gained compound can be had an impact, by taking albumin and IR-783 are compound as an example, regulate and control albumin concentration
When preparing IR-783@albumin complex from 2.5mg/ml to 40mg/ml, it is found that the reaction density of 40mg/ml produces largely
Precipitating assemble (as shown in Figure 4), and use 20mg/ml albumin reaction concentration below, when especially 20mg/ml, gained
Compound size is more satisfactory.Therefore in currently preferred preparation method, 1) the control solution A and solution B mixing in
The concentration of functional protein is in 2.5~20mg/ml afterwards, and more preferably control is in 20mg/ml.
The present inventor by experiment it has also been found that, in the preparation method, 1) described in functional protein and cyanine contaminate
The reaction ratio of material molecule also will affect the size of gained compound.Equally by taking the reaction of albumin and IR-783 as an example, two
Person's ratio is in the change procedure from 1:0.0625 to 1:2, it has been found that the ratio of 1:2 has slight precipitate to generate (Fig. 5 A), and ruler
Very little become larger (Fig. 5 B)；Therefore in currently preferred preparation method, 1) functional protein and the cyanine dye described in
Molecular ratios are preferably controlled in 1:1.
In addition, the incubation time after being blended for the functional protein described in 1) with cyanine dye molecule is to compound ruler
Very little influence, we carry out experimental verification, increase of the discovery with the reaction time, compound using two ratios of 1:0.5 and 1:1
The trend (as shown in Figure 6) increased is presented in size.Therefore in currently preferred preparation method, 1) the blending incubation time described in
It is 24 hours.
For in preparation method of the present invention 2) described in heating temperature, the present inventor also by Germicidal efficacy from room temperature,
50, the effect of compound after purification is handled under 60 to 70 degrees Celsius of different temperatures, finds room temperature and 50 degrees Celsius of processing
The complex stabilities that temperature is formed are poor, and dye molecule can be diffused out from compound；And 70 degrees Celsius of reaction temperature
Compound brightness (Fig. 9) is greatly reduced again.Therefore in currently preferred preparation method, 2) heating temperature described in is 55~
60 DEG C, most preferably 60 DEG C.
Further, the present invention also provides the compounds in 2nd area of near-infrared answering as fluorescent molecules imaging imaging agent
With.
In application scheme of the present invention, 2nd area of near-infrared is near infrared light of the wavelength at 900 nanometers or more
Wave band；It is preferred that 1000 nanometers or more of near infrared light wave band；More preferable 1100 nanometers or more of near infrared light wave band；Most preferably
1200 nanometers or more of near infrared light wave band.
In application scheme of the present invention, the fluorescent molecules imaging is included in various cells, tissue or other work
Imaging in body biology.
Existing two area's image probe of near-infrared based on individual molecule is low with quantum efficiency, living body disperse is very fast, steady
The qualitative weakness such as poor, regardless of all there is the imaging bottles that can not be solved in terms of small animal living body imaging or surgical navigational
Neck.Due to its shorter imaging window, it is necessary to carry out multiple injection administration and reach the visual purpose of tumor locus, but is this big
The multiple dosing of dosage, while also considerably increasing background signal, to make the low background in 2nd area of near-infrared and high-penetration depth
The big heavy discount of advantage.We carry out cyanine dye molecule with abundant existing albumin in vivo during the present invention proposes
Package is modified, and realizes the cyanine dye probe of albumin package for the first time, since cyanine molecule is coated by the stable ground of albumin,
On the one hand it since limitation vibration turns relaxation and enhancing intramolecular distortion charge transfer process, considerably increases 2nd area of near-infrared and shines
Efficiency；On the other hand circulation time in vivo is greatly strengthened by the composite construction that albumin wraps up, to reduce individual molecule
Disperse is very fast, weak tendency that stability is poor.During realizing albumin package cyanine dye molecule, we pass through optimization
Reaction condition realizes the blood vessel imaging of gained probe long circulating time, is obviously improved so that luminous and internal behavior be prepared
Two region probe system of near-infrared, be expected to be applied to subsequent two area's clinic navigating surgery of near-infrared in.
We demonstrate simultaneously, and the compound that other function protein and cyanine dye are formed in the same manner can be realized
The ideal effect of target tumor.And then our detailed comparisons its imaging effect in one area of near-infrared and near-infrared 2nd area, card
Cyanine compound, which is illustrated, has the imaging effect significantly improved in 2nd area of near-infrared.
Based on the solution of the present invention, following we further can also carry out DOTA chelate ring by the level-one amine to albumin
Covalence graft is realized double image probes of two areas of near-infrared/positron e mission computed tomography (PET), will be mentioned significantly
The accuracy of high clinical detection and surgical navigational.
In short, compound of the invention has than indocyanine green used in current surgical navigational, luminous efficiency is high, background
Small, the advantages that disperse is slower is scattered, the signal-to-noise ratio and penetration depth of living imaging will be greatly improved.
Detailed description of the invention
Fig. 1 is the schematic diagram of 4 kinds of reaction schemes described in embodiment 2-5.
Fig. 2 is reaction principle schematic diagram described in embodiment 2-5.
Fig. 3 A is the material object for the albumin complex that in embodiment 1 prepared by tri- dye molecule of IR-783, IR-12N3 and ICG
Figure.
Fig. 3 B is the dynamic for the albumin complex that in embodiment 1 prepared by tri- dye molecule of IR-783, IR-12N3 and ICG
Light scatters grain size distribution.
Fig. 4 is the present invention to the size distribution of reaction albumin concentration optimization process and product pictorial diagram.
Fig. 5 A is product pictorial diagram of the present invention for the optimization process of reaction feed ratio.
Fig. 5 B is product size distribution map of the present invention for the optimization process of reaction feed ratio.
Fig. 6 is the present invention to compound size distribution plot corresponding with the optimization in reaction time.
Fig. 7 is the composite molecular weight distribution map that in gel electrophoresis analysis embodiment 2-5 prepared by four kinds of reaction schemes.
Fig. 8 is the compound transmission electron microscope phenogram that in embodiment 2-5 prepared by four kinds of reaction schemes；Scheme two
(C2), the size of scheme three (C3) preparation is bigger compared to scheme one (C1), scheme four (C4).
Fig. 9 embodies influence of the treatment temperature for compound partial size in the present invention, and higher post-processing temperature can be made
Standby compound of the more stable and particle diameter distribution at 50-100 nanometers, the post-processing temperature of optimization are 60 degrees Celsius；
Figure 10 is living imaging recruitment evaluation of the embodiment 6 for the embodiment 2-3 IR-783@albumin complex prepared
Scheme (imaging effect figure that every group of two rows are respectively near-infrared one area and 2nd area in tri- groups of A, B and C), embodies the compound
Ideal blood vessel imaging ability.
Figure 11 is living imaging recruitment evaluation of the embodiment 6 for the embodiment 3-5 IR-783@albumin complex prepared
Figure, embodies the ideal blood vessel imaging ability of the compound and circulation time in vivo.
Figure 12 is that LED excites lower compound to the imaging effect figure of mouse thigh blood vessel in embodiment 7, it can be found that with
The red shift in section is imaged, imaging effect significantly improves.
Figure 13 is the assessment in embodiment 7 for mouse blood vessel imaging time window, it can be found that recording a video in 2nd area of near-infrared
In 30 minutes of record, blood vessel signal is remained intact, blood vessel with higher/muscle signal-to-noise ratio.
Figure 14 is that Complex Probes realize the near-infrared one of mouse Cerebral vascular image, two area imaging contrasts figure in embodiment 7.
Figure 15 is micro-imaging figure cerebrovascular for mouse in embodiment 8, compared to simple cyanine molecule, compound
With significantly improved Cerebral vascular image ability, as low as 3 microns of capillary can be imaged.
Figure 16 is the near-infrared that functional protein Erbitux (Erbitux) and the compound preparation of cyanine dye are selected in embodiment 9
Two area's combined probes, and then realize 2nd area of the targeting near-infrared imaging of SCC tumour.
Specific embodiment
Below with reference to embodiment, the present invention is further elaborated.
Cladding preparation of reagents used in each embodiment is as follows: preparing 40mg/ml (602 with phosphate buffer solution (PBS)
μM) bovine serum albumin(BSA) (BSA) solution, configure 26.7mM cyanine dye (ICG, IR-783 and IR-12N3) DMSO solution,
The glutathione solution containing 15%DMSO for configuring 0.25M, configures the glutaraldehyde solution of 0.25M.
Embodiment 1.
Three kinds of typical cyanine dye ICG, IR-783 and IR-12N3 are selected to respectively refer to 4 kinds of schemes that Fig. 1 is provided with white
Albumen cladding, concrete scheme are as follows:
500 microlitres of PBS are added in one: 500 microlitre of albumin solution of scheme, 11 microlitres of cyanine dyes are added, after mixing,
It reacts 24 hours in 37 degrees Celsius of concussion casees, is washed five times with the centrifugation filter membrane of 30k, post-processing temperature is 60 degrees Celsius 10 points
Clock.
500 microlitres of PBS are added in two: 500 microlitres of albumin solutions of scheme, 60 microlitres of glutathione solutions are added and mix；
11 microlitres of cyanine dyes are added to mix, reacts 24 hours in 37 degrees Celsius of concussion casees, is washed five times with the centrifugation filter membrane of 30k,
Post-processing temperature be 60 degrees Celsius 10 minutes.
500 microlitres of PBS are added in three: 500 microlitres of albumin solutions of scheme, 60 microlitres of glutathione and 24 microlitre penta is added
Dialdehyde solution mixes；11 microlitres of cyanine dyes are added to mix, are reacted 24 hours in 37 degrees Celsius of concussion casees, with the centrifugation of 30k
Filter membrane wash five times, post-processing temperature be 60 degrees Celsius 10 minutes.
500 microlitres of PBS are added in four: 500 microlitres of albumin solutions of scheme, 24 microlitres of glutaraldehyde solutions are added and mix；Add
Enter 11 microlitres of cyanine dyes to mix, reacts 24 hours in 37 degrees Celsius of concussion casees, washed five times with the centrifugation filter membrane of 30k, after
Treatment temperature be 60 degrees Celsius 10 minutes.
The equal clear of the compound of formation, having good stability, (as shown in Figure 3A, wherein C1, C2, C3 and C4 are respectively corresponded
Scheme one in Fig. 1 is to scheme four)；As shown in Figure 3B, wherein embodiment 2,3 prepare compound it is relatively large sized (referring to
The corresponding data point of GSH and GSH+GTD in abscissa), it was demonstrated that its multiple albumin, which fits together, forms 50-100 nanometers
Composite construction.Prove that the compound of four schemes preparation can be used as two region probe of near-infrared after tested.
Following emphasis is enumerated prepared by the present invention to illustrate using IR-783 as the embodiment for being wrapped by cyanine dye molecule
The performance characteristic and application advantage of compound.
2. simple blend of embodiment prepares the compound of IR-783 and albumin
Shown in preparation method such as Fig. 1 " scheme one ", specifically includes: IR- is added in the BSA solution of 40mg/ml (602 μM)
783 solution, control albumin and IR-783 reach the molar feed ratio of 1:1, mix, and control albumin reaction concentration and exist
20mg/ml is reacted 24 hours in 37 degrees Celsius of concussion casees, is washed five times with the centrifugation filter membrane of 30k, and post-processing temperature is taken the photograph for 60
Family name's degree 10 minutes, reaction process and gained composite structure can be found in the signal of Fig. 1,2, be in nanometer under transmission electron microscope
Graininess, as shown in Fig. 8 leftmost diagram.Gained compound is denoted as " IR-783@BSA ".
Glutathione reduction prepares the compound of IR-783 and albumin after embodiment 3. is blended
Shown in preparation method such as Fig. 1 " scheme two ", specifically includes: being added 60 in the BSA solution of 40mg/ml (602 μM)
The glutathione solution containing 15%DMSO of microlitre 0.25M adds IR-783 solution, and control albumin and IR-783 reach 1:
1 molar feed ratio mixes；And albumin reaction concentration is controlled in 20mg/ml, it is small that 24 are reacted in 37 degrees Celsius of concussion casees
When, washed five times with the centrifugation filter membrane of 30k, post-processing temperature be 60 degrees Celsius 10 minutes, reaction process and gained compound
Structure can be found in the signal of Fig. 1,2, be in Nanoparticulate under transmission electron microscope, such as Fig. 8 from left to right shown in second figure.Gained
Compound is denoted as " IR-783@BSA-GSH ".
Glutathione reduction and glutaraldehyde cross-linking prepare the compound of IR-783 and albumin after embodiment 4. is blended
Shown in preparation method such as Fig. 1 " scheme three ", specifically includes: being added 60 in the BSA solution of 40mg/ml (602 μM)
The glutaraldehyde solution of the glutathione solution and 24 microlitres of 0.25M containing 15%DMSO of microlitre 0.25M, it is molten to add IR-783
Liquid, control albumin and IR-783 reach the molar feed ratio of 1:1, mix；And control albumin reaction concentration in 20mg/ml,
It reacts 24 hours in 37 degrees Celsius of concussion casees, is washed five times with the centrifugation filter membrane of 30k, post-processing temperature is 60 degrees Celsius 10 points
Clock, reaction process and gained composite structure can be found in the signal of Fig. 1,2, be in Nanoparticulate under transmission electron microscope,
Such as Fig. 8 from left to right shown in third width figure.Gained compound is denoted as " IR-783@BSA-GSH-GTD ".
Glutaraldehyde cross-linking prepares the compound of IR-783 and albumin after embodiment 5. is blended
Shown in preparation method such as Fig. 1 " scheme four ", specifically includes: being added 24 in the BSA solution of 40mg/ml (602 μM)
The glutaraldehyde solution of microlitre 0.25M adds IR-783 solution, and control albumin and IR-783 reach the molar feed ratio of 1:1,
It mixes；And albumin reaction concentration is controlled in 20mg/ml, it reacts 24 hours in 37 degrees Celsius of concussion casees, is filtered with the centrifugation of 30k
Film washs five times, post-processing temperature be 60 degrees Celsius 10 minutes, reaction process and gained composite structure can be found in Fig. 1,2
Illustrate, is in Nanoparticulate under transmission electron microscope, as shown in the figure of the rightmost side Fig. 8.Gained compound is denoted as " IR-783@
BSA-GTD”。
We further carry out size table using compound of the gel electrophoresis for four reaction schemes of embodiment 2-5
Sign, discovery embodiment 3 use scheme two and embodiment 4 use scheme three produce biggish assembly (C2 in Fig. 7,
C3), this result is consistent with the dynamic light scattering data in Fig. 3 B with transmission electron microscope data (Fig. 8).
6. near infrared region living imaging recruitment evaluation of embodiment
We are composite I R-783@BSA, the IR-783@BSA-GSH, IR-783@BSA- for embodiment 2-5 preparation
GSH-GTD and IR-783@BSA-GTD carries out living body in-vivo imaging recruitment evaluation in near-infrared one, two area's imaging devices.
As shown in Figure 10, IR-783@BSA and IR-783@BSA-GSH compound is all demonstrated by preferable in-vivo imaging effect
Fruit.It is excreted faster with simple IR-783 molecule on the contrary, IR-783@BSA and IR-783@BSA-GSH is demonstrated by and mentions
High internal metabolism behavior.The imaging of 2nd area of near-infrared can more clearly differentiate mouse blood vessel, it was confirmed that it increases internal
Metabolism behavior.Meanwhile IR-783@BSA-GSH ratio IR-783@BSA has more preferably brightness and blood vessel imaging ability.
We further compare tri- kinds of BSA-GTD of@BSA-GSH-GTD and IR-783@of IR-783@BSA-GSH, IR-783 again
Close the in-vivo imaging behavior of object.We demonstrate that IR-783@BSA-GSH and IR-783@two kinds of compounds of BSA-GSH-GTD all have
Preferable brightness of image and circulation time in vivo (Figure 11), are suitble to a variety of imaging applications such as blood vessel.
7. mouse blood vessel imaging capability evaluation of embodiment
We select IR-783@BSA-GSH or IR-783@BSA-GSH-GTD compound further to assess its mouse blood vessel
Imaging capability.Due to the higher two area's quantum yield of near-infrared of compound, we can apply low power near-infrared LED light source
It is excited, realizes the mouse thigh blood vessel imaging preferably from one area of near-infrared to two area difference sections.As shown in figure 12, exist
Under the excitation of near-infrared LED, obtained excellent blood vessel imaging effect, and with the red shift in imaging subinterval, 1200 or
1300 nanometers of above band obtain extremely excellent blood vessel imaging ability.
We further pass through the video recording of 2nd area of near-infrared, have recorded the blood vessel imaging window injected after above two compound
Mouth time (Figure 13), finds in 30 minutes of video record, above two compound in the blood vessel protected well by signal
It holds, blood vessel/muscle signal-to-noise ratio can achieve 6 to 8.We demonstrate above two compound in the C57 mouse brain of shaving again
The blood vessel imaging ability of probe, as shown in figure 14, above two compound 2nd area of near-infrared reached the excellent cerebrovascular at
As ability, it is expected to be applied to subsequent cerebral vessels disease research and surgical navigational.
8. mouse blood vessel micro-imaging effect of embodiment
The Complex Probes of optimization are selected, we realize the cerebrovascular micro-imaging of mouse.At present near-infrared living body at
As being concentrated mainly on living body wide field equipment, and micro-imaging can fine structure in determination tissue definitely, thus real
Existing viviperception disease model.As shown in figure 15, the Mice brain tissues for having injected IR-783 BSA compound can realize high quality
Two area's tiny blood vessels of near-infrared imaging, as low as 3 microns of blood vessels can be recognized accurately.And in contrast, simple IR-783
Molecule can not accomplish similar imaging effect.
The compound of embodiment 9. other function protein and cyanine dye molecule
We utilize Erbitux (Erbitux) and cyanine dye referring to the method composite of embodiment 2, and preparation can target
The targeting compound of tumour EGFR receptor.We demonstrate the target in the nude mice model for being vaccinated with neck tumor (SCC)
The targeting ability (Figure 16) excellent to compound, although this targeting compound is absorbed with stronger liver, it still has
Certain molecular imaging application prospect.
Claims (14)
1. the compound of a kind of functional protein and cyanine dye molecule, it is by cyanine dye molecule and functional protein
By the compound being controllably self-assembly of, the compound outer layer is the functional protein, the functionality
Protein interior is enclosed with the cyanine dye molecule.
2. compound described in claim 1, it is characterised in that: the cyanine dye molecule is that have any one following bone
The derivative of frame structure:
Cyanine dye molecule further preferred ICG or IR-125 with the skeleton 1；
Cyanine dye molecule further preferred IR-820, IR-830 or Cy7.5 with the skeleton 2；
Cyanine dye molecule further preferred DiR or HITCI with the skeleton 3；
Cyanine dye molecule further preferred IRDye800cw, IR-783, IR12-N3, Cy7, IR- with the skeleton 4
775 or IR-780.
3. compound described in claim 1, it is characterised in that: the cyanine dye molecule be IR-783, IR-12N3 or
ICG。
4. compound described in claim 1, it is characterised in that: the functional protein is selected from albumin, glycoprotein, rouge
Any one in albumen, globulin or other antibody；The further preferred Erbitux of other antibody (Erbitux),
Anti-CD31 antibody or herceptin (herceptin).
5. compound described in claim 1, it is characterised in that: the compound is by the cyanine dye molecule and white
For albumen by what is be controllably self-assembly of, the compound outer layer is albumin, is enclosed with inside the albumin described
Cyanine dye molecule；The cyanine dye molecule is IR-783, IR-12N3 or ICG.
6. a kind of method for preparing compound described in claim 1, comprising:
1) solution A containing the functional protein is mixed with the solution B containing the cyanine dye molecule, is controlled
The functional protein and the cyanine dye molecule reach the molar ratio of 1:0.5~1, are blended and are incubated for 4-24 hours；
Intermediate product is obtained after purification；
2) 1) gained intermediate product 5~15 minutes are heated at 55 DEG C~70 DEG C, obtain the compound.
7. method of claim 6, it is characterised in that: 1) solution A described in further contain disulfide bond reducing agent and/or
Amino crosslinker；The ratio of the disulfide bond reducing agent and the functional protein is controlled in 40-60:1；The ammonia
The ratio of based cross-linker and the functional protein is controlled in 15~25:1；The preferred 2- sulfydryl of the disulfide bond reducing agent
Ethyl alcohol (beta-ME), dithiothreitol (DTT) (DTT) or reduced glutathione (GSH), most preferably reduced glutathione (GSH)；
The amino crosslinker preferably has the bifunctionality crosslinking agent of high reaction activity, most preferably glutaraldehyde or double succinyls with amino
Imines ester (double NHS).
8. method of claim 6, it is characterised in that: 1) functionality egg after the control solution A and solution B mix in
The concentration of white matter is in 2.5~20mg/ml, and more preferably control is in 20mg/ml.
9. method of claim 6, it is characterised in that: 1) functional protein and the cyanine dye molecule described in
Molar ratio is controlled in 1:1.
10. method of claim 6, it is characterised in that: 1) the blending incubation time described in is 24 hours.
11. method of claim 6, it is characterised in that: 2) heating temperature described in is 55~60 DEG C, most preferably 60 DEG C.
12. application of the compound described in claim 1 in 2nd area of near-infrared as fluorescent molecules imaging imaging agent.
13. application described in claim 12, it is characterised in that: 2nd area of near-infrared is wavelength at 900 nanometers or more
Near infrared light wave band；It is preferred that 1000 nanometers or more of near infrared light wave band；More preferable 1100 nanometers or more of near infrared light wave band；
Most preferably 1200 nanometers or more of near infrared light wave band.
14. application described in claim 12, it is characterised in that: the fluorescent molecules imaging is included in various cells, tissue
Or the imaging in other living body biologicals.
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